Establishment and Optimization of Culture Technique for Enteroids / 胃肠病学

Background:Enteroids are considered to be the best tool for studies on intestinal epithelium in vitro and have a widely application prospects,however,there are no associated reports in China. Aims:To establish and optimize the culture technique for enteroids and provide a fantastic platform for the basic research of small intestinal epithelial cells in China. Methods:L-WRN cells were cultured routinely and the conditioned medium with different concentrations of fetal bovine serum(FBS)was collected. Six to eight weeks old C57BL/ 6 mice were sacrificed and 15 cm small intestine from the terminal ileum was removed and cut longitudinally. Crypts were digested with EDTA and then collected and embedded in Matrigel? Matrix;after polymerization of Matrigel? Matrix,L-WRN conditioned medium at different concentration gradient was added. The budding ratio and length of buds were measured dynamically under microscope. The enteroids were re-embedded for subculture when certain length of buds was reached. Results:Compared with L-WRN conditioned medium containing 20% FBS,the conditioned medium containing 10% FBS was more favorable for enteroids culture in vitro. When conditioned medium accounted for 10% ,15% ,20% ,25% or 30% of the mixed medium,they all promoted the growth of enteroids and the 15% one seemed to yield better result. Conclusions:An enteroids culture technique was successfully established for the first time in China. When the L-WRN conditioned medium containing 10% FBS accounts for 15% of the mixed medium,it might promote budding better than the others.

Microarchitecture of the Palatine Tonsil in Sheep (Ovis aries) / Microarquitectura de la Tonsila Palatina en Oveja (Ovis aries)

The tissue pieces of palatine tonsil were collected from different postnatal age groups of sheep from the Corporation Slaughter House, Perambur, Chennai. The palatine tonsil consisted of a surface epithelium, capsule, tonsillar lobes, crypts, crypt epithelium and tonsillar follicles. The surface epithelium over the palatine tonsil was made up of non-keratinized stratified squamous epithelium in all the postnatal age groups studied. The palatine tonsil was clearly demarcated from the surrounding structures by a distinct connective tissue capsule and one septa dividing the tonsil into two lobes. The surface epithelium was invaginated into the substance of the tonsil to form primary and secondary crypts in each lobe. The crypt epithelium covered the regions of lymphoid follicles became lymphoepithelium. The macrophages were also observed in the epithelium. In the areas of lymphoepithelium the basement membrane was interrupted since lymphocytic infiltration was heavy into the epithelium. Numerous secondary tonsillar follicles with germinal centers separated by interfollicular areas were observed in the palatine tonsil. The tonsillar follicles consisted of a mantle zone, which was heavily populated with small darkly stained lymphocytes. These mantle zones were always oriented towards the crypts. The tonsillar follicles of young sheep showed many medium and small sized lymphocytes, lymphoblasts and also reticulocytes. The reticular cells usually appeared larger than lymphocytes and had a more abundant and organized cytoplasm with vacuoles.

Fueron recolectadas piezas de tejido desde la tonsila palatina de ovejas con diferentes edades postnatales, desde la Corporación Slaughter House, Perambur, Chennai. La tonsila palatina consistía en un epitelio de superficie, cápsula, lóbulos de las tonsilas, criptas, epitelio de las criptas tonsilares y folículos. El epitelio superficial sobre la tonsila palatina estaba compuesto, en todos los grupos estudiados, de epitelio escamoso estratificado no-queratinizado. La tonsila palatina se delimitó claramente de las estructuras circundantes por un tejido conectivo capsular y un septo dividiendo la tonsila en dos lóbulos. El epitelio superficial se invaginó dentro de de la tonsila para formar criptas primarias y secundarias en cada lóbulo. El epitelio de las criptas cubrió las regiones de folículos linfoides transfomándose en linfoepitelo. También se observaron macrófagos en el epitelio. En las áreas de linfoepitelo la membrana basal estaba interrumpida por la infiltración linfocitaria y fue mayor en el epitelio. Fueron observados numerosos folículos tonsilares secundarios con centros germinales separados por áreas interfoliculares. Los folículos tonsilares consistían en una zona del manto que estaba densamente poblada con pequeños linfocitos intensamente teñidos. Estas zonas del manto se orientaron siempre hacia las criptas. Los folículos tonsilares de las ovejas jóvenes mostraron muchos linfocitos de tamaño medianos y pequeños, linfoblastos y también reticulocitos. Las células reticulares usualmente aparecían más grandes que los linfocitos y tenían un citoplasma más abundante y organizado con vacuolas.

Neo-regeneration of urinary bladder: A desired metaplasia of autologous membrane from rectosigmoid colon containing stem cells of intestinal crypts.

The current management of diseases of urinary bladder requiring resection is by augmentation cystoplasty or transplantation of ureters. Transplantation of ureters is associated with morbidity and mortality. Ideal management will be by regenerating urinary bladder in vivo. Neo-regeneration of tissues and organs like abdominal wall, aponeurosis etc., has been attempted and patented. After neo-regeneration of mesoderm tissues and organs, regeneration of urinary bladder (developed from endoderm) was. In vivo surgical techniques were developed in dogs. It is known that the embryonic morphogenesis of urinary bladder is from uro-genital sinus of hind gut. A membrane, containing endoderm stem cells in crypts of recto-sigmoid colon, was surgically isolated and colonized with remnant of urinary bladder wall after extensive resection. Experimental study was performed in dogs, for 60 days to one and a half year. Regeneration of all the layers of tissues of the wall of urinary bladder was observed. The neo-regeneration phenomenon has been recognized as “desired metaplasia”. The regenerated neo tissue/organ on histological examination and cystometry studies was found compatible with normal urinary bladder. The hypothesis, neo-regeneration and desired metaplasia, is discussed.

Effect of melatonin on the induction of foci of aberrant crypts in the colon by azoxymethane in rats

<p><b>OBJECTIVE</b>A program of research was established on the question whether melatonin played a chemopreventive role in the development of foci of aberrant crypts in the intestinal tract of male rats. Male F344 rats were injected i.p. with an aqueous solution of 15 mg/kg azoxymethane (AOM) on day 50 and day 57, and a group was also injected i.p with 0.5 mg melatonin in 0.5 ml of 10% ethanol solution 5 times per week beginning at age 47 days. Foci and multiplicity of aberrant crypts were determined after 8 weeks. These groups of animals were kept in light daily from 4∶30 to 16∶30.</p><p><b>RESULTS</b>In the group receiving AOM and the melatonin injections, there were fewer foci of aberrant crypts in the colon and the average number of crypts was lower after 8 weeks, compared to the group on AOM alone.</p><p><b>CONCLUSIONS</b>Melatonin inhibited the formation of foci of aberrant crypts in the descending colon of rats. Also, it reduced the number of aberrant crypts per focus from foci with 3 and more aberrant crypts.</p>